Immunoglobulin complex comprising interleukin-15

ABSTRACT

Compositions, methods, and uses of recombinant immunoglobulin proteins, recombinant immunoglobulin protein complexes, carrier protein complexes, and a pharmaceutical composition including one or more of those to increase immune therapy effectiveness are presented. Preferred protein and protein complex comprise one or more functional moieties that includes a binding motif to a tumor-associated antigen, a T-cell activating molecule, and a chemokine. In some embodiments, the pharmaceutical composition includes two or more protein complexes, which are functionally distinct from each other. In other embodiments, the pharmaceutical composition includes a genetically-engineered microorganism including a first tumor-associated antigen and a T-cell activating molecule, a recombinant immunoglobulin protein complex, and a chemokine.

This application claims priority to U.S. Provisional application withthe Ser. No. 62/652,554, filed Apr. 4, 2018, which is incorporated byits entirety herein.

FIELD OF THE INVENTION

The field of the invention is immunotherapy, especially as it relates tocancer immune therapy with multiple treatment modalities.

BACKGROUND OF THE INVENTION

The background description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

All publications and patent applications herein are incorporated byreference to the same extent as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference. Where a definition or use of a term in anincorporated reference is inconsistent or contrary to the definition ofthat term provided herein, the definition of that term provided hereinapplies and the definition of that term in the reference does not apply.

Single small-molecule drug cancer treatments generally fail to provide acure, due to among other things, the high complexity of tumor biology.For the same reason, multi-drug treatment regimens tend to fail inremoving all cancer cells from a patient, and relapse is often simply aquestion of time. For example, it has become apparent that many tumorcells create a complex tumor microenvironment (TME) that typicallyincludes regulatory T cells (Tregs), myeloid derived suppressor cells(MDSCs), and tumor associated macrophages (TAMs) that prevent immunesurveillance by endogenous T cells and natural killer (NK) cells, reduceantigen presentation, and hinder the activity of adoptively transferredanti-tumor T cells (Front Surg 2016; 3:11; J Immunol 2008;181:5425-5432; or Semin Immunol 2016; 28:64-72).

Consequently, various attempts have been undertaken to modulate thetumor microenvironment to thereby enhance treatment effects. Forexample, US 2017/0087185 discloses the use of a lentiviral expressionsystem for the generation of genetically engineered monocytes andmonocyte-derived macrophages for immunotherapy. In US 2017/0231995,Bruton's tyrosine kinase (BTK) inhibitors are discussed to interferewith signaling between tumor cells and various immune competent cellswithin the tumor microenvironment. In yet another approach, as discussedin US 2014/0255341, therapeutic agents are used that increase localproduction of effector cell-attracting chemokines within a tumor, withconcomitant suppression of local production of chemokines that attractregulatory T(reg) cells. For example, such therapeutic agents includeToll-like receptor (TLR) agonists or other activators of NF-KB pathwayin combination with a blocker of prostaglandin synthesis or a blocker ofprostaglandin signaling, in combination with a type-1 interferon, or incombination with both a blocker of prostaglandin synthesis or signalingand with a type-1 interferon.

In addition, more attention has been grown to the use of chemokines thatattracts immune competent cell to the tumor microenvironment. Forexample, loss of chemokine CXCL14 was reported to be associated with lowinfiltration by dendritic cells, and overexpression of CXCL14 couldattract dendritic cell toward the tumor in vitro and in vivo (J Immunol2005; 174:5490-5498). Further, expression of CXCL14 by tumor cells couldalso increases natural killer (NK), CD4+ T, and CD8+ T cellinfiltration, implicating CXCL14 as a candidate of immune modulatoryreagents in the treatment regime for the cancer patient.

While those individual candidate treatment method, target and reagentsmay improve selected aspects of treatment, they still often fail to leadto complete remission of the tumor. Moreover, most of the knowntreatments may also have systemic effects due to the lack of specificityof action in the tumor microenvironment. Viewed from a differentperspective, all or almost all of the known treatments target only asingle aspect of tumor biology. Therefore, there remains a need forimproved compositions and methods to treat cancer using immune therapy.

SUMMARY OF THE INVENTION

The inventive subject matter is directed to various compositions of,methods for, and use of a recombinant protein or protein complex orpharmaceutical compositions including a recombinant protein or proteincomplexes to increase effectiveness of immune therapy in the cancerpatients. The recombinant protein or protein complex or pharmaceuticalcompositions includes a plurality of functional moieties that may affectthe tumor microenvironment from different aspects. Thus, one aspect ofthe inventive subject matter includes a recombinant immunoglobulinprotein complex that includes an Fc domain having two Fc portions, eachof which is coupled with a cytokine binding domain having a functionalmoiety. Each cytokine binding domain is coupled with a cytokine, andeach of the cytokine is coupled with a functional moiety. It isespecially preferred that at least two of the first, second, third, andfourth functional moieties are functionally distinct, and the first,second, third, and fourth functional moieties are selected from a groupconsisting of a binding motif to a tumor-associated antigen, a T-cellactivating molecule, and a chemokine.

Optionally, in this recombinant immunoglobulin protein complex, at leastone of the first and second cytokine binding domains is IL-15Rα, ormodified IL-15Rα that decreases interaction with IL-15Rβ or IL-15Rγ andcytokines binding to such cytokine binding domain is IL-15 or mutatedIL-15 such that the cytokine binding domain and cytokine of therecombinant immunoglobulin protein complex present strong IL-15 agonistfunction.

Also optionally, the binding motif to a tumor-associated antigen is anantibody, a portion of an antibody or single-chain variable fragment(scFv) that is specific to any tumor-associated antigen, preferably EGFRor its fragment thereof, or more preferably patient-specifictumor-specific neoepitope. With respect to T-cell activating molecule,T-cell activating molecule can be selected from a group consisting of anOX-40 ligand, a 4-1BB ligand, an OX-40 agonist antibody, a 4-1BB agonistantibody. A preferred chemokine may include CXCL-14.

In some embodiments, the recombinant immunoglobulin protein complex mayfurther include a fifth functional moiety that is coupled to N-terminusof at least one of the first and second Fc portions via a linker or thatis coupled at least one of the first cytokine binding domain or thefirst cytokine via a linker. In such embodiments, it is preferred thatthe linker is an acid-labile linker that can be cleaved in an acidictumor microenvironment. Alternatively and/or additionally, therecombinant immunoglobulin protein complex may further include a fifthfunctional moiety that is coupled to at least one of the first cytokinebinding domain or the first cytokine via a linker. In such embodiments,it is contemplated that the fifth functional moiety and the firstfunctional moiety are coupled to the first cytokine binding domain,wherein the linker comprises first and second sublinkers, and whereinthe first sublinker is coupled to the first functional moiety and thesecond sublinker is coupled to the fifth functional moiety.

The contemplated recombinant immunoglobulin protein complex may furtherbe coupled to a carrier protein via the Fc domain. Preferred carrierprotein can be selected from the following: protein A, protein G,protein Z, albumin, refolded albumin. Additionally, the carrier proteincan be further associated with an immune-stimulatory cytokine or achemokine that are coupled to the carrier protein via a linker.

In another aspect of the inventive subject matter, the inventorscontemplate a pharmaceutical composition that includes a plurality ofrecombinant immunoglobulin protein complexes. Each of the recombinantimmunoglobulin protein complexes includes an Fc domain having two Fcportions, each of which is coupled with a cytokine binding domain havinga functional moiety. Each cytokine binding domain is coupled with acytokine, and each of the cytokine is coupled with a functional moiety.It is especially preferred that at least two of the first, second,third, and fourth functional moieties are functionally distinct, and thefirst, second, third, and fourth functional moieties are selected from agroup consisting of a binding motif to a tumor-associated antigen, aT-cell activating molecule, and a chemokine. Preferably, thepharmaceutical composition includes at least three recombinantimmunoglobulin protein complexes, and the at least three recombinantimmunoglobulin protein complexes are functionally distinct from eachother.

Optionally, in this recombinant immunoglobulin protein complex, at leastone of the first and second cytokine binding domains is IL-15Rα, ormodified IL-15Rα that decreases interaction with IL-15Rβ or IL-15Rγ andcytokines binding to such cytokine binding domain is IL-15 or mutatedIL-15 such that the cytokine binding domain and cytokine of therecombinant immunoglobulin protein complex present strong IL-15 agonistfunction.

Also optionally, the binding motif to a tumor-associated antigen is anantibody, a portion of an antibody or single-chain variable fragment(scFv) that is specific to any tumor-associated antigen, preferably EGFRor its fragment thereof, or more preferably patient-specifictumor-specific neoepitope. With respect to T-cell activating molecule,T-cell activating molecule can be selected from a group consisting of anOX-40 ligand, a 4-1BB ligand, an OX-40 agonist antibody, a 4-1BB agonistantibody. A preferred chemokine may include CXCL-14.

In some embodiments, the recombinant immunoglobulin protein complex mayfurther include a fifth functional moiety that is coupled to N-terminusof at least one of the first and second Fc portions via a linker or thatis coupled at least one of the first cytokine binding domain or thefirst cytokine via a linker. In such embodiments, it is preferred thatthe linker is an acid-labile linker that can be cleaved in an acidictumor microenvironment. Alternatively and/or additionally, therecombinant immunoglobulin protein complex may further include a fifthfunctional moiety that is coupled to at least one of the first cytokinebinding domain or the first cytokine via a linker. In such embodiments,it is contemplated that the fifth functional moiety and the firstfunctional moiety are coupled to the first cytokine binding domain,wherein the linker comprises first and second sublinkers, and whereinthe first sublinker is coupled to the first functional moiety and thesecond sublinker is coupled to the fifth functional moiety.

The contemplated recombinant immunoglobulin protein complex may furtherbe coupled to a carrier protein via the Fc domain. Preferred carrierprotein can be selected from the following: protein A, protein G,protein Z, albumin, refolded albumin. Additionally, the carrier proteincan be further associated with an immune-stimulatory cytokine or achemokine that are coupled to the carrier protein via a linker.

Optionally, the pharmaceutical composition may further include a reagentupregulating CXCL-14 expression in a tumor cell, which may includehistone deacetylase (HDAC) inhibitor.

In still another aspect of the inventive subject matter, the inventorscontemplate a pharmaceutical composition that includes 1) agenetically-engineered microorganism that has a recombinant nucleic acidencoding a first tumor-associated antigen and a T-cell activatingmolecule, 2) a recombinant immunoglobulin protein complex, and 3) achemokine. The recombinant immunoglobulin protein complex includes an Fcdomain having two Fc portions, each of which is coupled with a cytokinebinding domain having a functional moiety, and cytokines that arecoupled to the cytokine binding domain. It is contemplated that at leastone of the first and second functional moieties is a binding motif to asecond tumor-associated antigen.

Optionally, the genetically-engineered microorganism is selected from agroup consisting of a virus, an yeast, a bacteria. Also optionally, therecombinant nucleic acid encodes a polytope. Also optionally, the firsttumor-associated antigen is a patient-specific and tumor specificneoepitope.

With respect to T-cell activating molecule, T-cell activating moleculecan be selected from a group consisting of an OX-40 ligand, a 4-1BBligand, an OX-40 agonist antibody, a 4-1BB agonist antibody. A preferredchemokine may include CXCL-14. Optionally, in this recombinantimmunoglobulin protein complex, at least one of the first and secondcytokine binding domains is IL-15Rα, or modified IL-15Rα that decreasesinteraction with IL-15Rβ or IL-15Rγ and cytokines binding to suchcytokine binding domain is IL-15 or mutated IL-15 such that the cytokinebinding domain and cytokine of the recombinant immunoglobulin proteincomplex present strong IL-15 agonist function.

In some embodiments, the first and second tumor-associated antigens aresame peptide antigens. Optionally, the second tumor-associated antigenis a patient-specific and tumor specific neoepitope and/or a portion ofEGFR.

Additionally and/or alternatively, the chemokine is CXCL-14, and/or thechemokine is coupled to the recombinant immunoglobulin protein complexvia a linker, preferably an acid-labile linker. In such embodiments, itis contemplated that the chemokine can be the at least one of the firstand second functional moieties and/or the chemokine is coupled to therecombinant immunoglobulin protein complex N-terminus of at least one ofthe first and second Fc portions.

Another aspect of the inventive subject matter includes a method ofenhancing immunotherapy or increasing effectiveness of immune therapy ina patient having a tumor. In this method, a recombinant immunoglobulinprotein including an Fc domain having two Fc portions, each of which iscoupled with a cytokine binding domain having a functional moiety isprovided. Each cytokine binding domain is coupled with a cytokine, andeach of the cytokine is coupled with a functional moiety. It isespecially preferred that at least two of the first, second, third, andfourth functional moieties are functionally distinct, and the first,second, third, and fourth functional moieties are selected from a groupconsisting of a binding motif to a tumor-associated antigen, a T-cellactivating molecule, and a chemokine. Then, the recombinantimmunoglobulin protein is administered to the patient in a dose andschedule effective to treat the tumor.

Optionally, in this recombinant immunoglobulin protein complex, at leastone of the first and second cytokine binding domains is IL-15Rα, ormodified IL-15Rα that decreases interaction with IL-15Rβ or IL-15Rγ andcytokines binding to such cytokine binding domain is IL-15 or mutatedIL-15 such that the cytokine binding domain and cytokine of therecombinant immunoglobulin protein complex present strong IL-15 agonistfunction.

Also optionally, the binding motif to a tumor-associated antigen is anantibody, a portion of an antibody or single-chain variable fragment(scFv) that is specific to any tumor-associated antigen, preferably EGFRor its fragment thereof, or more preferably patient-specifictumor-specific neoepitope. With respect to T-cell activating molecule,T-cell activating molecule can be selected from a group consisting of anOX-40 ligand, a 4-1BB ligand, an OX-40 agonist antibody, a 4-1BB agonistantibody. A preferred chemokine may include CXCL-14.

In some embodiments, the recombinant immunoglobulin protein complex mayfurther include a fifth functional moiety that is coupled to N-terminusof at least one of the first and second Fc portions via a linker or thatis coupled at least one of the first cytokine binding domain or thefirst cytokine via a linker. In such embodiments, it is preferred thatthe linker is an acid-labile linker that can be cleaved in an acidictumor microenvironment. Alternatively and/or additionally, therecombinant immunoglobulin protein complex may further include a fifthfunctional moiety that is coupled to at least one of the first cytokinebinding domain or the first cytokine via a linker. In such embodiments,it is contemplated that the fifth functional moiety and the firstfunctional moiety are coupled to the first cytokine binding domain,wherein the linker comprises first and second sublinkers, and whereinthe first sublinker is coupled to the first functional moiety and thesecond sublinker is coupled to the fifth functional moiety.

The contemplated recombinant immunoglobulin protein complex may furtherbe coupled to a carrier protein via the Fc domain. Preferred carrierprotein can be selected from the following: protein A, protein G,protein Z, albumin, refolded albumin. Additionally, the carrier proteincan be further associated with an immune-stimulatory cytokine or achemokine that are coupled to the carrier protein via a linker.

Still another aspect of the inventive subject matter includes a methodof enhancing immunotherapy or increasing effectiveness of immune therapyin a patient having a tumor. In this method, a pharmaceuticalcomposition including a plurality of recombinant immunoglobulin proteincomplexes is provided. Each of the recombinant immunoglobulin proteincomplexes includes 1) an Fc domain having first and second Fc portionscoupled with respective first and second cytokine binding domains havingrespective first and second functional moieties, 2) a first and secondcytokines coupled with a third and fourth functional moieties, where thefirst and second cytokine binding domains are coupled to. In thiscomposition, at least two of the first, second, third, and fourthfunctional moieties are functionally distinct, and the first, second,third, and fourth functional moieties are selected from a groupconsisting of a binding motif to a tumor-associated antigen, a T-cellactivating molecule, and a chemokine. Preferably, at least two of therecombinant immunoglobulin protein complexes functionally distinct.Then, the pharmaceutical composition is administered to the patient in adose and schedule effective to treat the tumor.

Optionally, the genetically-engineered microorganism is selected from agroup consisting of a virus, an yeast, a bacteria. Also optionally, therecombinant nucleic acid encodes a polytope. Also optionally, the firsttumor-associated antigen is a patient-specific and tumor specificneoepitope.

With respect to T-cell activating molecule, T-cell activating moleculecan be selected from a group consisting of an OX-40 ligand, a 4-1BBligand, an OX-40 agonist antibody, a 4-1BB agonist antibody. A preferredchemokine may include CXCL-14. Optionally, in this recombinantimmunoglobulin protein complex, at least one of the first and secondcytokine binding domains is IL-15Rα, or modified IL-15Rα that decreasesinteraction with IL-15Rβ or IL-15Rγ and cytokines binding to suchcytokine binding domain is IL-15 or mutated IL-15 such that the cytokinebinding domain and cytokine of the recombinant immunoglobulin proteincomplex present strong IL-15 agonist function.

In some embodiments, the first and second tumor-associated antigens aresame peptide antigens. Optionally, the second tumor-associated antigenis a patient-specific and tumor specific neoepitope and/or a portion ofEGFR.

Additionally and/or alternatively, the chemokine is CXCL-14, and/or thechemokine is coupled to the recombinant immunoglobulin protein complexvia a linker, preferably an acid-labile linker. In such embodiments, itis contemplated that the chemokine can be the at least one of the firstand second functional moieties and/or the chemokine is coupled to therecombinant immunoglobulin protein complex N-terminus of at least one ofthe first and second Fc portions.

Still another aspect of the inventive subject matter includes a methodof enhancing immunotherapy or increasing effectiveness of immune therapyin a patient having a tumor. In this method, a pharmaceuticalcomposition including at least two of 1) a genetically-engineeredmicroorganism that has a recombinant nucleic acid encoding a firsttumor-associated antigen and a T-cell activating molecule, 2) arecombinant immunoglobulin protein complex, and 3) a chemokine. Therecombinant immunoglobulin protein complex includes an Fc domain havingtwo Fc portions, each of which is coupled with a cytokine binding domainhaving a functional moiety, and cytokines that are coupled to thecytokine binding domain. It is contemplated that at least one of thefirst and second functional moieties is a binding motif to a secondtumor-associated antigen. Then, the pharmaceutical composition isadministered to the patient in a dose and schedule effective to treatthe tumor.

Optionally, the genetically-engineered microorganism is selected from agroup consisting of a virus, an yeast, a bacteria. Also optionally, therecombinant nucleic acid encodes a polytope. Also optionally, the firsttumor-associated antigen is a patient-specific and tumor specificneoepitope.

With respect to T-cell activating molecule, T-cell activating moleculecan be selected from a group consisting of an OX-40 ligand, a 4-1BBligand, an OX-40 agonist antibody, a 4-1BB agonist antibody. A preferredchemokine may include CXCL-14. Optionally, in this recombinantimmunoglobulin protein complex, at least one of the first and secondcytokine binding domains is IL-15Rα, or modified IL-15Rα that decreasesinteraction with IL-15Rβ or IL-15Rγ and cytokines binding to suchcytokine binding domain is IL-15 or mutated IL-15 such that the cytokinebinding domain and cytokine of the recombinant immunoglobulin proteincomplex present strong IL-15 agonist function.

In some embodiments, the first and second tumor-associated antigens aresame peptide antigens. Optionally, the second tumor-associated antigenis a patient-specific and tumor specific neoepitope and/or a portion ofEGFR.

Additionally and/or alternatively, the chemokine is CXCL-14, and/or thechemokine is coupled to the recombinant immunoglobulin protein complexvia a linker, preferably an acid-labile linker. In such embodiments, itis contemplated that the chemokine can be the at least one of the firstand second functional moieties and/or the chemokine is coupled to therecombinant immunoglobulin protein complex N-terminus of at least one ofthe first and second Fc portions.

In still another aspect of the inventive subject matter, the inventorscontemplate use of the pharmaceutical compositions or the recombinantimmunoglobulin protein complex described above for enhancingimmunotherapy or increasing effectiveness of immune therapy in a patienthaving a tumor.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments.

DETAILED DESCRIPTION

The inventors now discovered that the effectiveness of immune therapy,and particularly NK-cell or T-cell based immune therapy to a cancerpatient can be enhanced by specifically delivering a plurality ofanti-cancer drugs to the tumor microenvironment to so recruit NK or Tcells to the tumor microenvironment and/or induce tumor-specific NK or Tcell-mediated immune response.

Along the line, the inventors discovered that a recombinant proteincomplex or a pharmaceutical composition including a recombinant proteincomplex that has a plurality of functional moieties can be generated totreat the tumor. At least some functional moieties can be drugmolecules. As used herein, a drug molecule can be any molecule that cantreat a tumor to reduce the tumor growth, tumor cell activity,metastasis of the tumor, reduce immune suppression in the tumormicroenvironment, boost the effect of other cancer treatments, or thatcan monitor the progress or effect of other cancer treatment includingimmune therapy. For example, the drug molecule may be an anti-cancerdrug such that providing the anti-cancer drug in the tumormicroenvironment could boost effectiveness of the immune therapy in alocation-specific manner. In another example, the drug molecule may be amarker molecule such that the activity of the cytotoxic immune cells inthe tumor microenvironment can be determined by administering a modifiedimmune competent cell coupled with the marker molecule.

Thus, in an especially preferred aspect of the inventive subject matter,the inventors contemplate a recombinant immunoglobulin protein complexhaving a plurality of functional moieties, among which at least one ofthe functional moiety is configured to target the recombinantimmunoglobulin protein complex to the tumor microenvironment, and atleast another functional moiety is configured to induce T cell or NKcell mediated immune response in the tumor microenvironment. Suchmulti-functional recombinant protein is thought to increase theeffectiveness of the functional moieties, especially those inducesimmune response, by specifically directing them to the tumormicroenvironment, and even targeting them to a specific cell types inthe tumor microenvironment. As is discussed in more detail below, themulti-functional recombinant protein, in some aspects of the inventivesubject matter, may be based on a TxM scaffold or modified IL-15superagonist (ALT-803) (e.g., as shown inhttp://www.altorbioscience.com/our-science/il-15-protein-superagonist-and-scaffold-technology/)

As used herein, the term “tumor” refers to, and is interchangeably usedwith one or more cancer cells, cancer tissues, malignant tumor cells, ormalignant tumor tissue, that can be placed or found in one or moreanatomical locations in a human body. As used herein, the term “bind”refers to, and can be interchangeably used with a term “recognize”and/or “detect”, an interaction between two molecules with a highaffinity with a K_(D) of equal or less than 10⁻³M, 10⁻⁴M, 10⁻⁵M, 10⁻⁶M,or equal or less than 10⁻⁷M. As used herein, the term “provide” or“providing” refers to and includes any acts of manufacturing,generating, placing, enabling to use, or making ready to use.

In one exemplary and especially preferred aspect of the inventivesubject matter, the inventors contemplate a recombinant immunoglobulinprotein complex having a plurality of functional moieties. Mosttypically, the recombinant immunoglobulin protein complex comprises ormimics an ALT-803 or TxM structure. Of course, it is also contemplatedthat the recombinant immunoglobulin protein complex may also mimic aphenotype of human immunoglobulin A or M such that multiple recombinantimmunoglobulin protein complexes form a larger, multiple-unit, complex.

While it is preferred that the Fc portion is substantially a full sizeFc domain of a human IgG, IgM, IgE, or IgA for fully functional Fcdependent reaction (e.g., antibody-dependent cellular cytotoxicity(ADCC), etc.), it is also contemplated that Fc portion can be a fragmentof the full size Fc domain of human IgG, IgM, IgE, or IgA. Yet, it isespecially preferred that the Fc portion will include sufficientsequence to allow (i) formation of dimers via a disulfide bond, (ii)binding to protein A or protein G, and/or (iii) binding to the Sudlow-IIdomain of albumin, and especially refolded albumin (which may be loadedwith a drug such as a taxane). For example, each of the two Fc portionsincludes a hydrophobic interface to interact with each other to form adimer. The hydrophobic interface includes at least 10 amino acids, atleast 15 amino acids, at least 20 amino acids, at least 25 amino acids,similar to Fc domain of human immunoglobulin G. Alternatively, at leastone of the two Fc portions can be engineered such that the single Fcportion can be stable and soluble without forming a dimer with anotherFc portion.

Each of the Fc portion is typically coupled with a cytokine bindingdomain at the N-terminus of the Fc portion, and each of the cytokinebinding domain is bound to a ligand (a cytokine molecule). Any suitablecytokine binding domains are contemplated, including, but not limited tointerleukin-15 (IL-15) binding protein (e.g., a full length or an IL-15binding motif of IL-15 receptor α, etc.), CD25 (IL-2 binding protein),IL-4 receptor α, IL-13 receptor α, or IL-21 receptor α. Of course, thepreferred cytokine is the high affinity ligand for the cytokine bindingdomains, for example, IL-15 for a full length or an IL-15 binding motifof IL-15 receptor α, and IL-2 for CD25. In some embodiments, thecytokine binding domain is directly coupled to the N-terminus of Fcportion. In other embodiments, the cytokine binding domain is coupled tothe N-terminus of Fc portion via a linker or a spacer, which istypically between 3-30 amino acids, preferably between 5-20 amino acids,more preferably between 5-15 amino acids. The inventors contemplate thatglycine-rich sequences (e.g., gly-gly-ser-gly-gly, etc.) are preferredto provide structural flexibility between the Fc portion and thecytokine binding domain, especially when the cytokine binding domain isbulky and may provide steric hindrance to other nearby domains. Theinventors also contemplate that in some embodiments, at least one of thecytokine binding domains and cytokines coupled to those can besubstituted with other pairs of molecules coupled by high affinityprotein-protein interaction. For example, the pairs of molecules mayinclude an enzyme (preferably inactive enzyme)-peptide substrate, or atoxin receptor-inactive toxin (e.g., recombinant fusion toxin).

In some embodiments, the cytokine binding domain can be modified toreduce the biological effect of cytokine binding to the binding domain.For example, where the cytokine binding domain is IL-15 receptor α(IL-15Rα), and the cytokine is IL-15, the IL-15Rα:IL-15 complex cantrans-interact with a membrane-bound IL-15 receptor β and IL-15 receptorγ to elicit IL-15-mediated signaling cascade in the cell expressingIL-15 receptor β and IL-15 receptor γ. Thus, the inventors contemplatethat a portion of the IL-15Rα can be modified so that the interactionbetween the IL-15Rα:IL-15 and IL-15 receptor β or IL-15 receptor γ canbe prevented. For example, a portion of IL-15Rα critical for interactionwith IL-15 receptor β or IL-15 receptor γ may be removed or mutated suchthat the interaction can be abolished. In another example, an extrapeptide motif can be added to the IL-15Rα such that the recognition ofIL-15 receptor β or IL-15 receptor γ of IL-15Rα -IL-15 may beinterfered.

Alternatively and/or additionally, the cytokine can be modified toreduce such undesired effect. For example, where the cytokine is IL-15,IL-15 peptide can be modified to reduce the affinity to IL-15 receptor βor IL-15 receptor γ (e.g., deletion or substitution of amino acids ofIL-15 at positions 8, 61, 65, 72, 92, 101, 108, or 111, etc.). Otherexemplary mutations that may reduce the affinity of IL-15 to IL-15receptor β or IL-15 receptor γ are disclosed in U.S. Patent Pub. No.US2012/0177595, which is incorporated herein by reference in itsentirety.

The inventors contemplate that one or more cytokine binding domains andcytokines in the recombinant immunoglobulin protein complex, morepreferably each of the cytokine binding domains and cytokines, arecoupled with one or more functional moieties. While any desiredfunctional moieties are contemplated, in an especially preferredembodiment, at least one functional moiety is a binding motif to atumor-associated antigen, preferably a patient-specific and/ortumor-specific neoepitope. As used herein, the tumor-associated antigenrefers any antigen that can be presented on the surface of the tumorcells, which includes an inflammation-associated peptide antigen, atumor associated peptide antigen, a tumor specific peptide antigen, anda cancer neoepitope. Thus, a tumor-associated antigen can include anypeptide antigens and/or mutant peptide antigens that are specificallyexpressed on the tumor cells or over-expressed on the tumor cells. Forexample, the tumor-associated antigen may include a portion of epidermalgrowth factor receptor (EGFR), preferably, human epidermal growth factorreceptor 2 (HER2), CEA, MUC-1, CYPB1, PSA, brachyury, PD-L1, etc.Typically, the tumor associated antigens and neoepitopes (which aretypically patient-specific and tumor-specific) can be identified fromthe omics data obtained from the cancer tissue of the patient or normaltissue (of the patient or a healthy individual), respectively. Omicsdata typically includes information related to genomics,transcriptomics, and proteomics. As used herein, the cancer cells ornormal cells (or tissues) may include cells from a single or multipledifferent tissues or anatomical regions, cells from a single or multipledifferent hosts, as well as any permutation of combinations.

Any suitable binding motif (affinity portion) that can be linked to thecytokine binding domain (e.g., IL-15Rα of TxM) or cytokine (e.g., IL-15of TxM) of the recombinant immunoglobulin protein complex withoutproviding significant steric hindrance or functional defect and thatbinds specifically to tumor associated antigens and/or neoepitopes arecontemplated. Preferred binding motif includes a whole antibody (e.g.,IgG, IgM, IgE, and/or IgA), a portion of an antibody (e.g., one or moreFab, Fab′, F(ab)₂, etc.), or a single chain variable fragment (scFv).Alternatively, suitable affinity portions may also include proteins thatwere obtained by affinity maturation (e.g., using phage display) or byRNA display. In some embodiments, the protein complex may include one ormore binding motifs that target same tumor-associated antigen (e.g., twoscFv binding to Her-2, coupled to two cytokine binding domains,respectively). Alternatively and/or additionally, the protein complexmay include one or more binding motifs that target differenttumor-associated antigens (e.g., one scFv binding to Her2 that iscoupled to one cytokine binding domain and another scFv binding to apatient-specific tumor specific neoepitope that is coupled to anothercytokine binding domain, etc.). Alternatively and/or additionally, theprotein complex may include have mixed combination of one or morebinding motifs that target same tumor-associated antigen and one or morebinding motifs that target different tumor-associated antigens. Forexample, the protein complex may include first scFv binding to Her2 thatis coupled to one cytokine binding domain and second and third scFvsbinding to PD-L1 and a patient-specific tumor specific neoepitoperespectively that are coupled to another cytokine binding domain. Insuch embodiments, it is contemplated that the second and third scFvs arecoupled via a linker.

The inventors further contemplate that the recombinant immunoglobulinprotein complex also include an immune effector molecule, preferably amolecule that can induce T cell activation, that are coupled to at leastone of cytokine binding domain or cytokine of the protein complex. Anysuitable peptide, peptide fragment, and/or synthetic peptide that canincrease T cell activation at least 5%, at least 10%, at least 20%, atleast 30% are contemplated. Exemplary T cell activating moleculeincludes, but not limited to, an OX-40 ligand, a 4-1BB ligand, an OX-40agonist antibody, a 4-1BB agonist antibody. It is contemplated thatbinding of these exemplary T cell activating molecule to T cell maymimic the dendritic cell-T cell interaction to induce T cell activation.

Such contemplated protein complex that includes IL-15, tumor-associateantigen binding motif and T cell activating molecule are expected toprovide several notable advantages. Where the protein complex has anscFv that binds tumor associated antigen, the protein complex isspecifically targeted towards tumor cells in the tumor microenvironment.In addition, the presence of the IL-15 or IL-15 superagonist willadvantageously attract and activate T cells and NK cells, while thepresence of the Fc portion facilitates ADCC. Further, co-presence of Tcell activating molecule (e.g., OX-40 agonist antibody, a 4-1BB agonistantibody, etc.) can further augment T cell activation in the tumormicroenvironment where the T cells are more likely to interact withantigen presenting cells or tumor cells that present tumor-associatedantigens or neoepitopes such that more T cells can be activated in antumor-antigen specific manner.

In a further preferred embodiment, the inventors contemplate that therecombinant immunoglobulin protein complex also includes an immunestimulatory cytokine (e.g., IL-2, IL-8, etc.) and a chemokine (e.g.,CXCL14, CD40L, CCL2, CCL1, CCL22, CCL17, CXCR3, CXCL9, CXCL10, CXCL11,CXCL14, etc.), which can attract immune competent cells (e.g., T cells,B cells, NK cells, NKT cells, dendritic cells, macrophage etc.) to thetumor microenvironment and/or activate the immune competent cells.

In some embodiments, the cytokine binding domain or cytokine is directlycoupled to a functional moiety (e.g., via a cross-linker that uses thiolor amino groups, non-covalent coupling using hydrogen bonding orhydrophobic interactions, etc.). Alternatively and/or additionally, thecytokine binding domain or cytokine is coupled to a functional moietyvia a linker or a spacer, which is typically between 3-30 amino acids,preferably between 5-20 amino acids, more preferably between 5-15 aminoacids. Similar to coupling between the Fc portion and the cytokinebinding domain, glycine-rich sequences (e.g., gly-gly-ser-gly-gly, etc.)as a linker or a spacer are preferred to provide structural flexibilitybetween the cytokine binding domain (or cytokine) and the targetrecognition domain, especially when one or more target recognitiondomain is bulky and may provide steric hindrance to other targetrecognition domains.

Optionally, especially where the functional moiety is preferred to bereleased in the tumor microenvironment rather than being tethered to theprotein complex, the functional moiety can be coupled to one of thecytokine binding domain or cytokine via a linker that can be preferablycleaved in the tumor microenvironment. While any suitable linkers thatcan be preferentially cleaved in the tumor microenvironment and/or uponactivation of immune system are contemplated, one preferred linkerincludes a linker that is cleavable in a mild acidic environment (e.g.,at a pH between 3-6, at a pH between 4-6, at a pH between 4.5-5.5,etc.), yet stable in a neutral pH. For example, preferred acid-labilelinkers include a thimaleamic acid linker and an acid-cleavablehydrazine linker (e.g., hydrazine linker, etc.). It is contemplated thatany functional moieties including a drug molecule (either an anti-cancerdrug or a marker molecule) coupled to the immune competent cell via anacid-labile linker can be released in the mildly acidic tumormicroenvironment, such that the functional moieties can selectively andspecifically target the tumor microenvironment.

In some embodiments, a linker may comprise a plurality of sublinkersthat are connected to one support (e.g., similar to a dendrimerstructure), where the support is coupled to the immune competent cells.For the plurality of sublinkers, it is contemplated that at least twosublinkers have different linker types such that the linkers can becleaved or cannot be cleaved in different conditions. For example, thelinker may include an acid-labile linker that is conjugated with achemokine (e.g., CXCL14) and a glycine-rich linker that is conjugatedwith T cell activating molecule (e.g., OX-40 ligand). In such example,the chemokine can be cleaved and released when the immune competent cellenters the acidic tumor microenvironment, but the T cell activatingmolecule may stay conjugated in the protein complex in the samecondition. However, it is also contemplated that the at least twosublinkers are same type of linkers such that linkers can be cleaved inthe same condition.

While it is preferred that the functional moieties are coupled to eithercytokine binding domain or cytokine of the recombinant immunoglobulinprotein complex, it is also contemplated that one or more functionalmoieties can be coupled to Fc domain of the recombinant immunoglobulinprotein complex. In such embodiment, it is especially preferred that thefunctional moieties are coupled to the Fc domain via a cleavable linkersuch that ADCC mediated by Fc domain may not be prevented by functionalmoieties. Thus, one exemplary recombinant immunoglobulin protein complexmay be based on TxM that includes dimerized Fc domain, two IL-15 bindingdomain and two IL-15. Each of IL-15 binding domains is coupled with anscFv binding to HER-2 fragment via a glycine-rich linker, and each ofIL-15 is coupled to an OX-40 ligand and a 4-1BB ligand via adendrimer-type linker that includes two glycine-rich sublinkers that arecoupled to OX-40 ligand or a 4-1BB ligand, respectively. In each ofN-terminus of Fc portions of Fc domain, CXCL14 is coupled to the Fcportion via an acid-labile linker.

Additionally, the recombinant immunoglobulin protein complex can befurther coupled to a carrier molecule via Fc portion. Anypharmaceutically acceptable carrier molecules that can stably carry therecombinant immunoglobulin protein complex to the tumor microenvironmentare contemplated. Exemplary carrier molecules includes protein A,protein G, protein Z, albumin, refolded albumin, a nanoparticle (e.g.,quantum dots, gold nanoparticles, magnetic nanoparticles, nanotubes,polymeric nanoparticles, dendrimers, etc.), or a bead (e.g., polystyrenebead, latex bead, dynabead, etc.). Preferably, the nanoparticle and/orbeads have a dimension below 1 μm, preferably below 100 nm.

Alternatively and additionally, it is also contemplated that one or moreimmune-stimulatory cytokine or chemokine can be coupled to the carrierprotein via a linker such that the carrier molecule carries two distinctmolecules: 1) a recombinant immunoglobulin protein complex and 2) acytokine or a chemokine. In such embodiments, the immune-stimulatorycytokine or chemokine can be coupled to an anchor molecule that can beanchored to the carrier molecule directly or indirectly. For example,where the carrier protein is an albumin, the anchor molecule can be ahydrophobic peptide or glycolipids in any suitable size (e.g., in alength of at least 10 amino acids, 15 amino acids, 20 amino acids, 30amino acids, etc.) to fit in one of Sudlow's site I and II of thealbumin or any other hydrophobic area of the albumin.

In other aspect of the inventive subject matter, the inventorscontemplate a pharmaceutical composition that includes a plurality ofdistinct recombinant immunoglobulin protein complexes. As used herein,recombinant immunoglobulin protein complexes are considered distinct orfunctionally distinct where the recombinant immunoglobulin proteincomplexes have at least one different functional moiety. Thus, based onthe type of one different functional moiety that is not shared by tworecombinant immunoglobulin protein complexes, the recombinantimmunoglobulin protein complexes may or may not have a shared function.For example, the pharmaceutical composition may include a firstrecombinant immunoglobulin protein complex that includes two functionalmoieties: an scFv binding to HER2 and a chemokine CXCL14 (e.g., two scFvbinding to HER2 are coupled to two IL-15 binding domains (one scFv toone IL-15 binding domain) and two CXCL14 are coupled to two IL-15 (oneCXCL14 to one IL-15); and a second recombinant immunoglobulin proteincomplex that includes two functional moieties: an scFv binding to atumor neoepitope and an OX-40 ligand. In this example, two recombinantimmunoglobulin protein complexes have no shared functional moieties, yethave a shared function: recognizing or targeting a tumor cell. Inanother example, the pharmaceutical composition may include a firstrecombinant immunoglobulin protein complex that includes two functionalmoieties: an scFv binding to HER2 and a chemokine CXCL14; a secondrecombinant immunoglobulin protein complex that includes two functionalmoieties: an scFv binding to HER2 and an OX-40 ligand. In this example,the first and second recombinant immunoglobulin protein complexes have ashared functional moiety (scFv binding to HER2), yet functionallydistinct with two different functional moieties (CXCL14 and OX-40ligand).

While the numbers and types of recombinant immunoglobulin proteincomplexes in such pharmaceutical composition requires at least tworecombinant immunoglobulin protein complexes that are functionallydistinct, it is preferred that the pharmaceutical composition includesat least three recombinant immunoglobulin protein complexes that are allfunctionally distinct from each other. Yet, it is also contemplated thatwhere there are three or more than three recombinant immunoglobulinprotein complexes in the pharmaceutical composition, at least twocomplexes are functionally distinct, and the rest of them sharefunctions with at least one of the at least two complexes.

The plurality of recombinant immunoglobulin protein complexes can befurther coupled to a carrier molecule including protein A, protein G,protein Z, albumin, refolded albumin, a nanoparticle (e.g., quantumdots, gold nanoparticles, magnetic nanoparticles, nanotubes, polymericnanoparticles, dendrimers, etc.), or a bead (e.g., polystyrene bead,latex bead, dynabead, etc.). In some embodiments, the recombinantimmunoglobulin protein complexes are coupled to different portions ofthe carrier molecule (e.g., Sudlow I and Sudlow II sites of albumin orrefolded albumin, etc.) such that the carrier molecule can carry tworecombinant immunoglobulin protein complexes independently from eachother. In other embodiments, recombinant immunoglobulin proteincomplexes are coupled to the carrier molecule via an anchor molecule. Insuch embodiments, it is also contemplated that the anchor moleculeincludes a dendrimer-type linker to couple two recombinantimmunoglobulin protein complexes independently or separately via twosublinkers (e.g., cleavable or non-cleavable).

In addition to CXCL14 as a functional moiety of the recombinantimmunoglobulin protein complex, or as an alternative thereof, thepharmaceutical composition may also include one or more reagentupregulating endogenous CXCL-14 expression in a tumor cell. Exemplaryreagents include some anti-cancer drug such as 5-aza-2′-deoxycytidine(decitabine or dacogen) or histone deacetylase (HDAC) inhibitor (e.g.,trichostatin A, etc.). Alternatively, the pharmaceutical composition canbe accompanied with a radiation therapy or chemotherapy that mayincrease CXCL-14 expression in a tumor cell.

In still another aspect of the inventive subject matter, the inventorscontemplate that a pharmaceutical composition can be generated toinclude three elements: 1) a genetically-engineered microorganism thatincludes or expresses tumor-associated antigen and a T-cell activatingmolecule, 2) a recombinant immunoglobulin protein complex having one ormore functional moieties, and 3) a chemokine. Preferably, themicroorganism is genetically engineered to express tumor-associatedantigen and a T-cell activating molecule as two separate and distinctpeptides. For example, the microorganism includes a recombinant nucleicacid having at least two nucleic acid segments (a sequence element): afirst nucleic acid segment encoding a tumor-associated antigen and asecond nucleic acid segment encoding a T-cell activating molecule in asingle reading frame such that two nucleic acid segments can betranslated into a single protein having two peptide segments under thesame promoter. In this case, the inventors contemplate that the firstand second nucleic acid segments are spaced with a spacer sequence(e.g., a nucleic acid sequence encoding a linker or a spacer of at least10 amino acids, 15 amino acids, 20 amino acids, etc.). In otherembodiments, the two nucleic acid segments may be transcribed separatelyinto two distinct peptides. In still other embodiments, the two nucleicacid segments are present in the same reading frame, but separated bynucleic acid sequences encoding a type of 2A self-cleaving peptide (2A).As used herein, 2A self-cleaving peptide (2A) refers any peptidesequences that can provide a translational effect known as “stop-go” or“stop-carry” such that two sub-segments in the same mRNA fragments canbe translated into two separate and distinct peptides. Any suitabletypes of 2A peptide sequences are contemplated, including porcineteschovirus-1 2A (P2A), thosea asigna virus 2A (T2A), equine rhinitis Avirus 2A (E2A), foot and mouth disease virus 2A (F2A), cytoplasmicpolyhedrosis virus (BmCPV 2A), and flacherie virus (BmIFV 2A).

It is especially preferred that the recombinant peptides encoded by therecombinant nucleic acid are expressed or present in the antigenpresenting cells such that the antigen presenting cells expresses thetumor associated antigen and the T cell activating molecule on thesurface to induce antigen-specific T cell activation. Thus, therecombinant nucleic acid is further placed in an expression vector to sodeliver the recombinant nucleic acid to the antigen-presenting cells(e.g., dendritic cells, etc.), or to transcribe the nucleic acidsequence in bacteria or yeast so that the recombinant peptide encoded bythe nucleic acid sequence can be, as a whole, or as fragments, deliveredto the antigen presenting cell. Any suitable expression vectors that canbe used to express protein are contemplated. Especially preferredexpression vectors may include those that can carry a cassette size ofat least 1 k, preferably 2 k, more preferably 5 k base pairs.

Thus, in one embodiment, the microorganism is a virus, and a preferredexpression vector includes a viral vector (e.g., nonreplicatingrecombinant adenovirus genome, optionally with a deleted ornon-functional E1 and/or E2b gene). In still further embodiments, themicroorganism is a bacteria, and the expression vector can be abacterial vector that can be expressed in a genetically-engineeredbacterium, which expresses endotoxins at a level low enough not to causean endotoxic response in human cells and/or insufficient to induce aCD-14 mediated sepsis when introduced to the human body. One exemplarybacteria strain with modified lipopolysaccharides includes ClearColi®BL21(DE3) electrocompetent cells. This bacteria strain is BL21 with agenotype F- ompT hsdSB (rB- mB-) gal dcm lon λ(DE3 [lacI lacUV5-T7 gene1 ind1 sam7 nin5]) msbA148 ΔgutQΔkdsD ΔlpxLΔlpxMΔpagPΔlpxPΔeptA. In thiscontext, it should be appreciated that several specific deletionmutations (ΔgutQ ΔkdsD ΔlpxL ΔlpxMΔpagPΔlpxPΔeptA) encode themodification of LPS to Lipid IV_(A), while one additional compensatingmutation (msbA148) enables the cells to maintain viability in thepresence of the LPS precursor lipid IVA. These mutations result in thedeletion of the oligosaccharide chain from the LPS. More specifically,two of the six acyl chains are deleted. The six acyl chains of the LPSare the trigger which is recognized by the Toll-like receptor 4 (TLR4)in complex with myeloid differentiation factor 2 (MD-2), causingactivation of NF-κB and production of proinflammatory cytokines. LipidIV_(A), which contains only four acyl chains, is not recognized by TLR4and thus does not trigger the endotoxic response. While electrocompetentBL21 bacteria is provided as an example, the inventors contemplates thatthe genetically modified bacteria can be also chemically competentbacteria. Alternatively, or additionally, the microorganism is a yeast,and the expression vector can also be a yeast vector that can beexpressed in yeast, preferably, in Saccharomyces cerevisiae (e.g.,GI-400 series recombinant immunotherapeutic yeast strains, etc.).

The recombinant immunoglobulin protein complex in this pharmaceuticalcomposition may include at least one functional moiety, preferably twomoieties. While the functional moieties can be at least one of a bindingmotif to a tumor-associated antigen, a T cell activating molecule and/ora chemokine, it is preferred that at least one functional moiety is abinding motif to a tumor-associated antigen such that the recombinantimmunoglobulin protein complex can be targeted to the tumor cells in thetumor microenvironment. In some embodiments, the tumor-associatedantigen targeted by the recombinant immunoglobulin protein complex maybe same with the tumor-associated antigen that is expressed in theantigen presenting cells by the genetically-engineered microorganism. Inother embodiments, the tumor-associated antigen targeted by therecombinant immunoglobulin protein complex may be different from thetumor-associated antigen that is expressed in the antigen presentingcells by the genetically-engineered microorganism. For example, thetumor-associated antigen targeted by the recombinant immunoglobulinprotein complex can be a tumor-specific, patient-specific neoepitope,while the tumor-associated antigen that is expressed in the antigenpresenting cells is HER2.

It is further contemplated that the pharmaceutical composition includesan immune stimulatory cytokine (e.g., IL-2, IL-8, etc.) and a chemokine(e.g., CXCL14, CD40L, CCL2, CCL1, CCL22, CCL17, CXCR3, CXCL9, CXCL10,CXCL11, CXCL14, etc.), which can attract immune competent cells (e.g., Tcells, B cells, NK cells, NKT cells, dendritic cells, macrophage etc.)to the tumor microenvironment and/or activate the immune competentcells. In some embodiments, the immune stimulatory cytokine or achemokine can be coupled with the recombinant immunoglobulin proteincomplex. In other embodiment, the immune stimulatory cytokine or achemokine can be coupled with the recombinant immunoglobulin proteincomplex can be coupled to the surface of the genetically-modifiedmicroorganism via a cleavable linker (e.g., acid-labile linker). In suchembodiments, the linker can be conjugated to theN-hydroxysuccinimidyl-PEG (PEG-NHS), by which the linker covalentlybonds with all kinds of membrane proteins having amino groups on cellsurfaces. Alternatively, the linker can be conjugated with PEG to formPEG-glycolipid or with poly(vinyl alcohol) carrying alkyl side chains(PVA-alkyl) such that the conjugated linker can anchor to the membranelipid bilayer of the genetically modified microorganism (e.g., virus)through hydrophobic interactions. In still other embodiments, the immunestimulatory cytokine or a chemokine can be present in the pharmaceuticalcomposition as a distinct molecule (not directly coupled to therecombinant immunoglobulin protein complex).

Additionally, where the chemokine is coupled to the surface ofgenetically-engineered microorganism, it is also contemplated that therecombinant immunoglobulin protein complex can be also coupled to thesurface of the genetically-engineered microorganism. In this embodiment,it is preferred that the recombinant immunoglobulin protein complex andthe chemokine are coupled to the genetically-engineered microorganismvia cleavable linker, preferably acid-labile linker such that therecombinant immunoglobulin protein complex and chemokine can beseparated from the genetically engineered organism once the complex(genetically-engineered microorganism with recombinant immunoglobulinprotein complex and chemokine) is placed in the acidic tumormicroenvironment. Alternatively, the pharmaceutical composition havingthree elements as described above is further coupled to a carrierprotein (e.g., protein A, protein G, protein Z, albumin, refoldedalbumin) via a linker (cleavable or non-cleavable) such that allelements can be targeted to the tumor microenvironment together.

Such generated recombinant immunoglobulin protein complex and/orpharmaceutical compositions that includes recombinant immunoglobulinprotein complex(es) can be formulated in any pharmaceutically acceptablecarrier (e.g., as a sterile injectable composition) and administered toa patient having a tumor to increase effectiveness of immune therapy toso treat the tumor (e.g., to modulate (e.g., reduce, abrogate, etc.)immune suppression by the tumor, to reduce the tumor size, etc.). Insome embodiments, the recombinant immunoglobulin protein complex and/orpharmaceutical compositions can be administered via systemic injectionincluding subcutaneous, subdermal injection, or intravenous injection.In other embodiments, where the systemic injection may not be efficient(e.g., for brain tumors, etc.) or more localized treatment is desired,it is contemplated that the recombinant immunoglobulin protein complexand/or pharmaceutical compositions can be administered via intratumoralinjection. As used herein, the term “administering” refers to bothdirect and indirect administration of the compounds and compositionscontemplated herein, where direct administration is typically performedby a health care professional (e.g., physician, nurse, etc.), whileindirect administration typically includes a step of providing or makingthe compounds and compositions available to the health care professionalfor direct administration.

With respect to dose and schedule of the formulation administration, itis contemplated that the dose and/or schedule may vary depending on thetype of protein, protein complex, or the type of the pharmaceuticalcomposition (e.g., virus, bacteria, yeast, in combination withrecombinant protein complex, etc.), a type and prognosis of disease(e.g., tumor type, size, location), health status of the patient (e.g.,including age, gender, etc.). While it may vary, the dose and schedulemay be selected and regulated so that the formulation does not provideany significant toxic effect to the host normal cells, yet sufficient tobe reduce immune suppression by reduced CXCL-14 expression or presencein the tumor microenvironment. Thus, in a preferred embodiment, anoptimal or desired condition of administering the formulation can bedetermined based on a predetermined threshold. For example, thepredetermined threshold may be a predetermined local or systemicconcentration of CXCL-14 in the tumor microenvironment. Therefore,administration conditions are typically adjusted to have CXCL-14increased in the tumor microenvironment at least 20%, at least 30%, atleast 50%, at least 60%, at least 70% at least for 24 hours, 48 hours,72 hours, 7 days, etc. In another example, the predetermined thresholdmay be a predetermined local or systemic concentration of cytokine(e.g., IFN-γ, IL-10, IL-13, etc.) released from activated NK cells orconcentration of T cell activating cytokines (e.g., IL-12, IL-23, IL-1b,IL-6, TGF-β, etc.) that are expressed by T cells upon interaction ofantigen presenting cells to differentiate into subset of activated Tcells. Therefore, administration conditions are typically adjusted tohave the concentration of cytokine increased at least 20%, at least 30%,at least 50%, at least 60%, at least 70% at least locally orsystemically. Moreover, it is contemplated that the compounds andcompositions presented herein may be co-administered (contemporaneouslyor sequentially) with NK cells. For example, suitable NK cells includeautologous NK cells as well as NK92 cells and derivatives thereof (e.g.,aNK cells, haNK cells, taNK cells, al commercially available fromNantKwest, 9920 Jefferson Blvd. Culver City, Calif. 90232).

Without wishing to be bound by any specific theory, the inventorscontemplate that administration of pharmaceutical composition (therecombinant immunoglobulin protein complex, a recombinant proteincomplex having a carrier protein, a plurality of recombinant proteincomplex associated with a carrier, a pharmaceutical composition having agenetically-engineered microorganism and a recombinant protein complex,etc.) to a patient is expected to cause the delivery of thepharmaceutical composition in a tumor microenvironment, preferably bytargeting a tumor cell. When the composition is delivered, it isexpected that multiple functional moieties of the composition willprovide distinct but complementary or even synergistic effect to thetumor microenvironment. For example, where the recombinant proteincomplex having three functional moieties, 1) a binding motif to atumor-associated antigen, 2) a T-cell activating molecule, and 3) achemokine coupled to TxM backbone, it is expected that the recombinantprotein complex will target the tumor microenvironment via binding tothe tumor-associated antigen expressed on the tumor cell. In the tumormicroenvironment, the chemokine (e.g., CXCL14) will increase theinfiltration of immune competent cells including dendritic cells, andthe T cell activating molecule (e.g., OX-40 ligand) will induce T cellactivation by binding to T cell receptor (OX-40) and mimicking dendriticcell-T cell interaction. In addition, the presence of the IL-15 or IL-15superagonist will advantageously attract and activate T cells and NKcells, while the presence of the Fc portion facilitates ADCC. Thus, inthis example, administration of the recombinant protein complex can notonly overcome the suppression of antitumor immune response bydownregulation of CXCL14, but also further boost the antitumor immuneresponse by attracting and activating immune competent cells in thetumor microenvironment. The inventors contemplate that similarcombinatorial effect can be obtained by administering a pharmaceuticalcomposition that has a plurality of recombinant protein complexes,which, in combination, introduce a binding motif to a tumor-associatedantigen, a T-cell activating molecule, a chemokine, IL-15 superagonist,and Fc domains.

In another example, where the pharmaceutical composition includes agenetically-engineered microorganism including a tumor-associatedantigen and a T-cell activating molecule, a recombinant immunoglobulinprotein complex with a functional moiety, and chemokine, it is expectedthat the pharmaceutical composition will be target the tumormicroenvironment by binding to the tumor-associated antigen expressed onthe tumor cell. Where the genetically-engineered microorganism is avirus, the virus will infect the antigen presenting cells to so producethe tumor-associated antigen and the T-cell activating molecule in theantigen presenting cells. Presentation of tumor-associated antigen andthe T-cell activating molecule (e.g., OX-40 ligand) will induceactivation of T cells that are tumor-associated antigen-specific(recognizing tumor-associated antigen) by binding to T cell receptor(OX-40). In addition, release of chemokine (e.g., CXCL14) will increasethe infiltration of immune competent cells including dendritic cells,and the presence of the IL-15 or IL-15 superagonist in the recombinantimmunoglobulin protein complex will advantageously attract and activateT cells and NK cells, while the presence of the Fc portion in therecombinant immunoglobulin protein complex will facilitates ADCC.

Thus, it should be appreciated that the compositions and methodspresented herein can provide more effective treatment using multipleanti-tumor compounds by specifically targeting the multiple compounds tothe tumor microenvironment and concurrently delivering of the compoundsto a small target area such that the amount and the type of multipleanti-tumor compounds can be co-controlled. It is expected that suchconcurrent and targeted delivery of multiple anti-tumor compounds wouldsynergize the effects of multiple anti-tumor compounds compared toindividual delivery of the anti-tumor compounds, where the deliverytiming, location, and effect of the individual anti-tumor compound mayvary. Further, such approach would also provide an effective focaltreatment to some tumor cells that are affected by specifictumor-associated pathway. For example, EGFR overexpression in the tumorcell may downregulate CXCL14 expression that results in increased immuneresistance of the tumor cells. Thus, targeted delivery of CXCL14 andother immune activating molecules (e.g., OX-40 ligands, etc.) to thetumor environment with EGFR binding motif as a functional moiety of theprotein complex can reverse immune resistance of the tumor cellsoverexpressing EGFR.

In addition, the inventors contemplate that the compositions presentedherein, especially the recombinant immunoglobulin protein complex canmimic the function of antigen presenting cells by binding to the tumorcells expressing a tumor-associated molecules via a binding motif to thetumor-associated antigen. Once bound to the tumor-associated molecule onthe surface of the tumor cell, the recombinant immunoglobulin proteincomplex can activate T cell-mediated immune response against the tumorcells via T-cell activating molecule (e.g., OX-40 ligand) associatedwith recombinant immunoglobulin protein complex. Optionally, release ofchemokine (e.g., CXCL14) from recombinant immunoglobulin protein complexwill increase the infiltration of immune competent cells to furtherboost the immune response. Thus, the recombinant immunoglobulin proteincomplex acts like a dendritic cells (like an avatar) expressing tumorassociated molecule on its surface to induce immune response against thetumor cell. Consequently, such activation of immune system may revertthe immune-suppressive environment of tumor microenvironment toimmune-active environment, which can be especially beneficial to treat apatient having an immune-suppressed tumor.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the scope of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. As used in the description herein and throughoutthe claims that follow, the meaning of “a,” “an,” and “the” includesplural reference unless the context clearly dictates otherwise. Also, asused in the description herein, the meaning of “in” includes “in” and“on” unless the context clearly dictates otherwise. Where thespecification claims refers to at least one of something selected fromthe group consisting of A, B, C . . . and N, the text should beinterpreted as requiring only one element from the group, not A plus N,or B plus N, etc.

What is claimed is:
 1. A recombinant immunoglobulin protein complex,comprising: an Fc domain having first and second Fc portions coupledwith respective first and second cytokine binding domains havingrespective first and second functional moieties, wherein the first andsecond cytokine binding domains are IL-15Rα; a first cytokine coupledwith a third functional moiety and a second cytokine coupled with afourth functional moiety, wherein first and second cytokines are IL-15;wherein the first cytokine binding domain is coupled with the firstcytokine and the second cytokine binding domain is coupled with thesecond cytokine; and wherein at least two of the first, second, third,and fourth functional moieties are functionally distinct, and the first,second, third, and fourth functional moieties are selected from a groupconsisting of a T-cell activating molecule, a chemokine, and ananti-tumor-associated antigen binding motif, wherein the T-cellactivating molecule is OX-40L, wherein the chemokine is CXCL-14, andwherein the tumor associated antigen is EGFR.
 2. The protein complex ofclaim 1, wherein the first and second Fc portions form a dimer.
 3. Theprotein complex of claim 1, further comprising a fifth functional moietythat is coupled to N-terminus of at least one of the first and second Fcportions via a linker.
 4. The protein complex of claim 3, wherein thelinker is an acid-labile linker.
 5. The protein complex of claim 1,further comprising a fifth functional moiety that is coupled to at leastone of the first cytokine binding domain or the first cytokine via alinker.
 6. The protein complex of claim 5, wherein the fifth functionalmoiety and the first functional moiety are coupled to the first cytokinebinding domain, wherein the linker comprises first and secondsublinkers, wherein the first sublinker is coupled to the firstfunctional moiety and the second sublinker is coupled to the fifthfunctional moiety, and wherein at least one of the sublinkers is anacid-labile linker.
 7. The protein complex of claim 1, wherein theprotein complex is coupled with a carrier protein via the Fc domain, andwherein the carrier protein is selected from the following: protein A,protein G, protein Z, albumin, refolded albumin.
 8. A pharmaceuticalcomposition comprising: a plurality of recombinant immunoglobulinprotein complexes, each of the recombinant immunoglobulin proteincomplexes comprising: an Fc domain having first and second Fc portionscoupled with respective first and second cytokine binding domains havingrespective first and second functional moieties, wherein the first andsecond cytokine binding domains are IL-15Rα; a first and a secondcytokine coupled with a third and a fourth functional moieties,respectively, wherein first and second cytokines are IL-15; wherein thefirst and second cytokine binding domains are coupled with the first andsecond cytokines, respectively; wherein at least two of the first,second, third, and fourth functional moieties are functionally distinct,and the first, second, third, and fourth functional moieties areselected from a group consisting of a binding motif to atumor-associated antigen, a T-cell activating molecule, and a chemokine,wherein the T-cell activating molecule is OX-40L, wherein the chemokineis CXCL-14, and wherein the tumor associated antigen is EGFR.
 9. Thecomposition of claim 8, wherein the first and second Fc portions form adimer.